Disposable reactor module and detection system

ABSTRACT

A disposable reactor module, monitoring/optical detection system and related hardware for, inter alia, chemical reactions including Polymerase Chain Reactions.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to the field of devices for performingchemical and/or bio-chemical reactions under a temperature-controlledenvironment. More particularly, the present invention relates to adevice for real-time monitoring/detecting of Polymerase Chain Reaction.

Analytical processes that only require small amounts of DNA have manyapplications in various fields, such as microbiology, forensics, foodscience, bio-defense, and water purification. Another application ofsuch processes is for pre-implantation genetic diagnosis (PGD) wherethere is only one cell to work with and to extract DNA from. PGDrequires an answer quickly so that the embryos can be selected totransfer back without having to freeze them.

Polymerase chain reaction (PCR) is a very valuable technique, becausethe reaction is highly specific, and capable of creating large amountsof copied DNA fragments from minute amounts of samples, for bothsequencing and genotyping applications. For this reason, PCR has wideapplications in clinical medicine, genetic disease diagnostics, forensicscience, and evolutionary biology. Recently, miniaturized PCR deviceshave attracted great interest because they have many advantages overconventional PCR devices, such as portability, higher thermal cyclingspeed, and significantly reduced reagents/sample consumption. Mostmini/micro PCR devices can be classified into two types, static chamberPCR chips and dynamic flow PCR chips.

The first type of device uses stationary thermal cyclers to heat andcool a static volume of liquid in a micro-chamber. In these devices,either the micro-chamber is manufactured separately and placed incontact with an external heater, or the micro-chamber and themicro-heater are bonded together to form a complete microchip. Aportable PCR device has been described with specially designed ceramicheaters and the corresponding PCR tubes by Belgrader et al. [P.Belgrader et al., Analytical Chemistry, 73, 286 (2001)]. In their work,the PCR reaction was achieved in a very short time period but the totalreaction volume was still as large as conventional PCR. The micro PCRsystem designed by Yang et al. [J. Yang et al., Lab on a Chip, 2, 179(2002)] controlled the temperature of a micro PCR reactor by two Peltierthermoelectric devices sandwiching the reactor. Because heat sinks andfans are attached to the Peltier thermoelectric devices for betterthermal management, it is difficult to operate the PCR and access thePCR chip after the installation. Lin et al [Y. C. Lin et al., Sensorsand Actuators B: Chemical, 71, 127 (2000)] use a PCR system with areaction well fabricated in a silicon wafer sealed with a glasssubstrate and place a heater at the bottom of the silicon wafer. In thisdesign, a small reaction volume is used to improve the temperatureuniformity. However, it is difficult to fill and collect the PCRsolution through the two holes on the top cover. Nagai et al. [H. Nagaiet al., Analytical Chemistry, 73, 1043 (2001)] pattern micro-chambers ofvarying sizes onto silicon wafers and run the PCR using a commercialthermal cycler. PCR chips with a reaction chamber and a micro-heaterpatterned onto a silicon wafer using micro-fabrication technologies arealso widely used in other PCR works to speed up the heating and coolingprocesses during the PCR cycles. Because of the integrated micro-heaterand temperature sensor, all chips are fabricated using photolithography,metal film deposition, etching, and oxidation processes, etc. Thus, theyare very expensive unless the chips are fabricated in high volumeproduction. Giordano et al. [B. C. Giordani et al., AnalyticalBiochemistry, 291, 124-132 (2001)] focuses an infrared light onto apolyimide chip and heats a small volume of PCR sample very quickly.However, the infrared heating system is complicated and increases theoperation cost greatly.

The second type of device, a dynamic flow-through PCR device, heats andcools PCR reactants by flowing the reactants through differenttemperature zones. A typical flow-through thermal cycler is one withthin film platinum heaters and sensors patterned onto a silicon wafer togenerate three different temperature zones. A flow-through thermalcycler using thermal convection flow also exists. Another flow-throughPCR chip pumps the reagents between three reaction chambers using abi-directional peristaltic pump. PCR reactions are also achieved in acontinuous flow mode by pumping in a ring chamber with controlledtemperature regions. Compared to the first type of PCR device, theflow-through type can reduce the heating and cooling time and thusshorten the total time of PCR reaction. However, it is difficult toexamine the PCR results and to collect the PCR product in the secondtype of PCR system. Reliability of this type of device cannot be assuredunless reliable pumping and inter-channel connection are available at anacceptable cost.

Research has also been done towards integrating PCR with either pre-PCRor post PCR processes to further utilize the advantages ofmicrofluidics. Real-time PCR, as it is known, is highly attractivebecause it can detect and quantify PCR results through real-timeanalysis of fluorescent signals generated during the reaction, withoutthe conventional post-PCR processes such as gel electrophoresis.

In real-time PCR [Bassler, H. A. et al. The use of a fluorogenic probein a PCR-based assay for the detection of Listeria monocytogenes. Appl.Environ. Microbiol. 61 (1995) 3724-3728; Livak, K. J. et al.Oligonucleotides with fluorescent dyes at opposite ends provide aquenched probe system useful for detecting PCR product and nucleic acidhybridization. PCR Methods Appl. 4 (1995) 357.362], a reporterfluorescence dye and a quencher dye are attached to an oligonucleotideprobe. Negligible fluorescence from the reporter dye's emission isobserved once both dyes are attached to the probe. Once PCRamplification begins, DNA polymerase cleaves the probe, and the reporterdye is released from the probe. The reporter dye, which is separatedfrom the quencher dye during every amplification cycle, generates asequence-specific fluorescent signal. Real-time PCR detection is basedon monitoring the fluorescent signal intensity produced proportionallyduring the amplification of a specific PCR product (e.g., an E. coliDNA); therefore, it is a direct and quantitative method with highsensitivity. Such a method has been used to detect E. coli Shiga-liketoxin genes in ground beef [Witham P. A., Yamashiro, C. T., Livak, K. J.and Batt, C. A., A PCR based assay for the detection of Escherichia coliShiga-like toxin genes in ground beef. Appl Environ Microbiol1996;62:1347-1353].

Real-time technologies have been applied by using FAM dye conjugatedprobes (a fluorescence dye; there is 5-FAM, 6-FAM and 5/6-FAM, its fullname is 5-carboxyfluorescein or 6-carboxyfluorescein) and SYBR greendyes. Through these processes, the real-time PCR reactions are conductedin customized flat polypropylene tubes with optical windows forfluorescence detection, the reaction volume ranging from 25 μL to 100μL. The requirement of a large amount of DNA template limits theseapplications. There also exists a miniature spectrometer capable ofdetecting a spectrum of fluorescence by using DNA labeled SYBR greendye. However, this system uses a commercial capillary thermal cycler.The overall system does not differ very much from conventional real-timePCR systems.

While real-time PCR has significant advantages compared to regular PCR,there are limitations to the application of real-time PCR techniques.During real-time PCR, the optical detection system must monitor thefluorescence intensity in real time. At least two separate sets ofexcitation-detection wavelength pairs must be available at each PCR wellto identify both the desired and control species in each well. As thenumber of wells and/or desired light interaction increases, the opticalinfrastructure grows greatly, increasing the complexity, cost, and sizeof the optical detection module.

Currently, the instruments for conducting real-time PCR are bulky andexpensive, and are only available in a few large hospitals and majormedical centers. Therefore, there is a need to develop an improvedsystem that will allow this valuable technique to be more widely used.

SUMMARY OF THE INVENTION

In general, applications that involve detecting gene mutations,detecting bacteria and viruses, performing genetic testing, or the like,can be performed using the present invention. These applications can befound in the fields of microbiology, forensics, food science, waterpurification, etc. For the purpose of this description, the inventionwill be described specifically with respect to PCR, but should not belimited to that application. The present invention can be used withother various applications, such as Enzyme Linked Immuno Sorbent Assay(ELISA), which is a sensitive immunoassay that uses an enzyme linked toan antibody or antigen as a marker for the detection of a specificprotein, especially an antigen or antibody. It is often used as adiagnostic test to determine exposure to a particular infectious agent,such as the AIDS virus, by identifying antibodies present in a bloodsample.

The present invention provides a miniature device consisting of areactor module made of a combination of glass and polymer and used witha miniature thermal cycler to perform real-time and regular PCR.Compared to silicon or glass PCR chips, the present device does not needmicromachining or photolithography processes. The fabrication of thereactor modules of the invention is very simple and low in cost. Thesereactor modules are disposable after a single use. This can avoid thepotential of contamination associated with other non-disposable PCRreactor modules due to reuse of the reaction chamber. In one embodiment,the present device fits a standard fluorescence microscope and thus itis possible to do real-time PCR tests using this system without anelaborate and expensive real-time PCR machine. This can make a real-timePCR test affordable to most biomedical laboratories by using theirexisting fluorescence microscopes. The present device is flexible interms of the sample volume and the number of wells that can be changedaccording to the applications.

In addition, the present invention also provides a fluorescencedetection system to establish a stand-alone real-time PCR system. Thedevice may be made of a small enough size to be portable.

In accordance with a first broad aspect of the present invention, thereis provided a disposable reactor module comprising: a non-reflective,thermally conductive substrate; and a layer of polymer on the substrate,the layer of polymer having at least one reaction well for receiving afluid sample, the polymer being chemically inert, non-adherent to DNA,and reacting in a stable manner to heating and cooling.

In accordance with a second broad aspect of the present invention, thereis provided a miniature multiplex fluorescence detection system fordetecting fluorescence emissions from at least one sample on a reactormodule having a plurality of reaction wells, the system comprising: atleast one light source coupled to the reaction wells, for generatinglight at excitation wavelengths; at least one detector for receivingdetection wavelengths from the reaction wells; and, an optical switchingdevice, coupled between the detector and the reaction wells on thesubstrate, to direct emissions of fluorescence to the detector.

In accordance with a third broad aspect of the present invention, thereis provided a method for real-time monitoring/detecting of atemperature-controlled chemical reaction involving fluorescenceemissions, the method comprising: providing at least one fluid sample ina disposable reactor module comprising a non-reflective, thermallyconductive substrate and a layer of polymer on the substrate, the layerof polymer having at least one reaction well for receiving the sample,the polymer being chemically inert, non-adherent to DNA, and reacting ina stable manner to heating and cooling; sealing at least one reactionwell; heating and cooling the reactor module to allow the chemicalreaction to progress in the at least one reaction well; directingexcitation wavelengths to the sample to cause fluorescence emissions;capturing the fluorescence emissions from the sample; and monitoring thechemical reaction by processing the fluorescence emissions.

In accordance with a fourth broad aspect of the present invention, thereis provided a system for real-time monitoring of a chemical reactioninvolving fluorescence emission-detection, the system comprising: adisposable reactor module, a sealant, a miniature multiplex fluorescencedetection system for detecting fluorescence emissions from the sampleson the reactor module having reaction wells, and a control module forcontrolling the fluorescence detection system and monitoring thechemical reaction by processing the fluorescence emissions. The reactormodule comprises: a non-reflective, thermally conductive substrate; anda layer of polymer on said substrate, the layer of polymer havingreaction wells for receiving fluid samples, the polymer being chemicallyinert, non-adherent to DNA, and reacting in a stable manner to heatingand cooling. The sealant prevents evaporation of the fluid samplecontained in the reaction wells of the reactor module. The fluorescencedetection system comprises: at least one light source coupled to thereaction wells, for generating light at excitation wavelengths; at leastone detector for receiving detection wavelengths from the reactionwells; and a fiber optical switching device, preferably corresponding tothe number of reaction wells, coupled between the detector and thereaction wells on the substrate, to direct emissions of fluorescence tothe detector. A heating and cooling module modulates the temperature ofthe samples, and a stage receives the reactor module and couples thereactor module to the heating and cooling module.

In one embodiment, the control module is connected to both the miniaturereactor module and the fluorescence detection system. It controls andsynchronizes the operation of the reactor module and the opticaldetection system. Alternatively, the fluorescence detection system isconnected to a computer that will externally process the fluorescenceemissions and monitor the chemical reaction.

In accordance with a fifth broad aspect of the invention, there isprovided a device for real-time monitoring/detecting of atemperature-controlled chemical reaction involving fluorescenceemission-detection, the device comprising: a miniature multiplexfluorescence detection system for detecting fluorescence emissions fromsamples contained in the reaction wells of a reactor module, the systemcomprising: at least one light source coupled to the reaction wells, forgenerating light at excitation wavelengths; at least one detector forreceiving detection wavelengths from said reaction wells; an opticalswitching device, coupled between said detector and the reaction wells,to direct emissions of fluorescence to said detector; a heating andcooling module for modulating a temperature of said samples; a stagecoupled to said heating and cooling module for receiving said reactormodule; and, a control module for controlling the fluorescence detectionsystem and monitoring the chemical reaction by processing thefluorescence emissions.

In one embodiment, the control module is connected to both the miniaturereactor module and the fluorescence detection system. It controls andsynchronizes the operation of the reactor module and the opticaldetection system. Alternatively, the fluorescence detection system isconnected to a computer that will externally process the fluorescenceemissions and monitor the chemical reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 is a cross-sectional view of the layer structure of the PCR chip;

FIG. 2 is a top view of the PDMS layer of the reactor module with fourreactant wells;

FIG. 3 is a flowchart of the fabrication of the casting molds;

FIGS. 4A and 4B illustrate masks for the single-well and four-well PCRchips, respectively;

FIG. 5 is a schematic of the PCR chip installation with external forces;

FIG. 6 is a cross-sectional view of the layer structure of the PCR chipwith an additional chip substrate layer;

FIG. 7 is a diagram illustrating the system of one embodiment of thepresent invention;

FIG. 8 is a schematic diagram of the optical detection module inaccordance with an exemplified embodiment;

FIG. 9 is a diagram illustrating the working principle of the filtercube;

FIG. 10 is a graph of the fluorescence intensity of three runs ofreal-time PCR having different initial DNA templates;

FIG. 11 is a graph of the fluorescence intensity of three runs ofreal-time PCR having different volumes of mixture, but the same initialDNA concentration;

FIG. 12 is the gel electrophoresis results of 3 μl and 7 μl PCR mixturereactions;

FIG. 13 is a graph of the fluorescence intensity from a three-wellreactor module where each well has the same DNA template amount;

FIG. 14 is a graph of the fluorescence intensity from a three-wellreactor module with different template DNA concentrations in each well;and

FIG. 15 is a graph of Fluorescence intensity curves obtained by fiberoptical detection system from real-time PCR of 150 bp E. coli O157:H7stx 1 DNA.

FIG. 16 illustrates the actual cycling temperature in the well of asingle-well reactor module in an experiment for E. coli O157:H7 stx1PCR.

FIG. 17 illustrates the results of PCR tests with different DNA usingthe chip system compared to a commercial PCR machine.

FIG. 18 illustrates the results of PCR tests with genomic DNA (2054)under different conditions with the chip system.

FIG. 19 shows schematically the assembly of a multiple-well reactormodule in accordance with one embodiment of the invention.

It will be noted that throughout the appended drawings, like featuresare identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The reactor module in accordance with one aspect of the presentinvention is used with a heating and cooling module. In one embodiment,the heating and cooling module is a miniature thermal cycler. In theexamples described herein where fluorescence is being monitored, withthe exception of the example described with reference to FIG. 15, thisreactor module was placed on the stage of a standard fluorescencemicroscope, and the reaction was monitored using the fluorescencemicroscope.

From hereon, the reactor module combined with the heating portion of theheating and cooling module will be referred to as a PCR chip. The PCRchip is illustrated in FIG. 1. Its overall dimension is shown in FIG. 2,which shows an example of a four-well chip. A single-well chip has thesame overall dimensions, such as width, length and height. The onlydifference is the location of the well, as well as the structures aroundthe well. The heater sits on a Teflon substrate 40, which can be fixedon the device chassis. PCR wells 30 are built in a PDMS layer 34, sit onthe glass substrate 36, and are covered by a thin glass microscope coverslip 32 (0.1˜0.2 mm). Then, these three layers, which in one embodimentof the invention form reactor module 48, sit on top of the heater 38.The heater 38 can be purchased from Omega (Model No. KHLV-101/10). Theglass substrate 36 can be a commercial micro cover glass/coverslip(size: 22×22 mm).

While PDMS was chosen as the best material, other polymers such as PMMA(Polymethylmethacrylate) can be used. A person skilled in the art willreadily identify that any material that is chemically inert,non-adherent to DNA, optically transparent, and reacts to heating andcooling in a stable manner can be used instead of PDMS. The advantage ofusing a cheap plastic like PDMS is that there is no micro-machining orlithographic process involved to make the wells, and therefore theoverall costs of production are negligible. The reactor module itselfbecomes disposable and issues of contamination involved in cleaning andreusing this apparatus are no longer a problem.

Theoretically, a reactor module is a simple structure and can be madeeasily by constructing wells to contain PCR agents. However, there aregreat challenges in the design and fabrication of the reactor modulewhen a miniature PCR chip is expected to be able to operate at a generalcondition, such as thirty cycles of denature (30 seconds), extension (30seconds) and annealing (30 seconds) at each cycle. In one embodiment,the reactor module was fabricated using the PDMS casting, cutting andbonding techniques as described below.

The PDMS mold is manufactured using a soft lithography technique.Masters containing the desired chip pattern are made by spin coatingSU-8 negative photoresist on a glass slide to a nominal thickness of 25μm. The final thickness is decided by controlling the speed of thespinning coat machine. The relationship between the thickness and thespeed can be further referred to in the data sheet for SU-8-25photoresist provided by MicroChem Inc. The photoresist film is thenhardened through a two stage direct contact pre-exposure bake procedure(65° C. for 5 min and 95° C. for 15 min) and exposed to UV light for 10seconds through a transparency mask containing the desired chip pattern.A two-stage post-exposure bake procedure (65° C. for 1 min 95° C. for 2min) is then used to enhance cross-linking in the exposed portion of thefilm. The slide is then placed in quiescent developer solution for 8 to12 min to dissolve the unexposed photoresist, leaving a positive reliefcontaining the chip pattern. Liquid PDMS is then poured over the masterand cured at 65° C. for 6 to 12 h yielding a negative cast of the chippattern (Generally, 10:1 PDMS and cure agency are used, but it was found15:1 PDMS and cure agency give better results). In the cured PDMS withthe chip pattern, through-holes are punched to form the reaction wellwhen the PDMS layer is bonded with a glass plate. A thin layer of glassis used to cover the reactor module of the PCR chip after providing thereaction agents in the reaction wells and sealing the reaction well tokeep the reaction agents from leaking out of the well.

The process for the master fabrication using SU-8 goes in steps as shownin FIG. 3, referring to the SU-8-25 datasheet provided by MicroChem. Inthe substrate pre-treat step, the glass substrate is soaked in acetonefor half an hour (or the clean glass slides are stored in acetone beforecoating with SU-8 photoresist), is heated on the hot plate for half anhour and then is treated in the plasma cleaner for 2 minutes. The glasssubstrate is coated with SU-8-25 by using a spin coater, which is set torun at 500 rpm for 5 seconds and at 1200 rpm for 20 seconds.

The two masks illustrated in FIGS. 4A and 4B are used to develop the PCRchips for single-well and four-well structures, respectively. Thesemasks are used to create desired PDMS casting mold structures using theSU-8 negative photoresist. In FIGS. 4A and 4B, the white areas representthe transparent areas in the mask. This means that the white areasrepresent protrusive parts in the cast mold and the grooves in thereactor modules fabricated using the mold. Thus, in both the single-wellstructure (FIG. 4A) and the four-well structure (FIG. 4B), there aregrooves around the reaction wells. These grooves around the reactionwells are used to enhance the sealing effect at the interface betweenthe PDMS layer 34 and top cover glass 32. The grooves or spacerssuccessfully break down the leakage links, provide a space for enfoldingany bubbles which may form in the reaction well 30 during the heatingprocess, and then avoid further spreading of the leaking gap. Thedimension of the well 30 is decided based on the requirements of theamount of PCR reaction agent, as well as the overall size of the PCRchip. Preferably, the dimensions of the well provide for a volume offrom about 0.1 μl to about 50 μl so that a sample of from about 0.1 μlto about 50 μl can be contained within the well. However, the volume ofthe well can be either larger or smaller to accommodate the givenreaction.

A micro cover glass 32 deposited with a thin PDMS film is alsofabricated for better bonding between the thin glass 32 and the PDMSlayer 34. The coating process is conducted using the spin coat machine,which is set to operate at 500 rpm for 5 seconds and 300 rpm for 20seconds. This process results in a layer of PMDS film with thickness ofaround 20 to 30 μm. This thin PDMS layer not only improves the bondingresults but also strengthens the thin glass and prevents it frombreaking.

It was observed that the liquid in the wells 30 of the PCR chip driedout faster because of the bubbles generated in the wells when the chipis heated up. Some of them were dry within four or five cycles. This isa great challenge for the real PCR process because most of the PCRshould run around 30 cycles to ampliiy the DNA to sufficient amounts.Liquid starts to leak out at the interfaces between the PDMS and glasscover. Since bubbles are generated at the high temperature, they pushliquids out of the wells through any tiny gaps between the PDMSsubstrate and the glass cover. Therefore, a good seal of the PCR well isrequired to make the chip withstand the whole PCR process without beingdried out. It was found that the structure w the grooves and/or thespacers about the wells of the present invention can withstand thethirty cycles at the required temperature profile for each cycle.

In both of the single well structure and the four-well structure,grooves around the reaction well were used to enhance the sealing effectat the interface between the PDMS layer 34 and top glass cover 32. Thiscover breaks down the leakage links and then avoids further spreading ofthe leaking gap. The size of the grooves depend on the well size. Thecritical parameter is the thickness of the groove. The thicker thegroove is, the better the sealing is. However, the thermal performanceof the chip decreases if the thickness of the groove is too much.Ideally, the groove thickness should range from 40 micron to 500 micron.Although the self-sealing characteristics of the PDMS surface make itpossible to seal the interface between the PDMS layer 34 and thePDMS-coated glass cover 32 to some extent, the different thermalexpansion coefficients for the different materials, such as PDMS, glass,and reaction agents, result in different deformations in each materialand thus the reaction agents may leak from the reaction wells. It isworse if bubbles are generated within the liquid when it is heated up.Therefore, to enhance the seal between the PDMS layer 34 and the glasscover 32, a mechanism, as shown in FIG. 5, was used to stop the leakagefrom the reaction wells by applying external forces. The PCR chip isplaced on a support stage 42 and force is applied as shown to provideproper sealing. It was observed that this kind of installation helps insolving the leakage problems. However, for four-well cases, not all fourwells can be sealed perfectly if the force applied over the chip surfaceis non-uniform.

As an alternative or addition to the grooves, a spacer may be placed onthe upper surface of the polymer layer. The spacer may be of aring-type, polygonal, or comparable design and surrounds the peripheryof at least one well. The peripheral spacer functions as to alleviatethe problems of leakage of the sample out of the reaction wells byproviding an open space at the topmost portion of the well for gaseousfluids including bubbles to exit from the liquid reagent. Preferably thespace provides for an additional 50 μm to about 500 μm in height thoughmay be larger or smaller depending on the specific reaction. Also, mostoften the glass cover 32 is utilized with the peripheral spacer and inconjunction provides an enclosed volume for the accumulation of evolvedgases from within the reaction well.

In an additional embodiment, the bubble problems are further resolved byan alternative cover for the reactor module of the PCR chip. Instead ofusing the full microscope cover slip to cover the reactor module (22mm×22 mm), a quarter size of the original cover slip is used to onlycover the small area above and around the PCR well in the reactormodule. The smaller size of the glass cover can significantly reduce theleaking effects of the uneven surface of the fabricated reactor modulesince it is very difficult to obtain a completely flat and smoothreactor module surface with the current fabrication conditions.

Also alternatively, instead of a glass cover, a layer of mineral oil isprovided on top of the sample in each of the reaction wells. This isused to prevent evaporation of the sample and also avoids the bubbleproblem. Other types of oil, such as silicon oil, can also be used. Anytype of transparent, non-aqueous, unreactive solution having arefractive index close to the fluid in the sample so as not to cause anydistortion effects, and having a high boiling point may be used as asealant.

To easily adapt to different PCR thermal conditions required bydifferent DNAs, the embodiment of the PCR chip, as shown in FIG. 1, ismodified into a further embodiment as shown in FIG. 6. In thisembodiment, a flat and smooth surface for a chip support substrate 44can make it easier to hold the PCR chip without interrupting the filmheater 38 position and provide a uniform thermal resistant condition atthe interface between the reactor module 48 and heater 38. It can alsoprevent the chip from cracking, usually caused by irregularities in thesurface of the reactor module 48. On the other hand, a temperaturesensor, such as a thermocouple, can be put into the support substrate 44to monitor the system temperature and thus control the thermal conditionapplied to the PCR chip.

Thermal cycling can be affected by the sizes of the reaction wells 30and the reactor module 48 itself, as well as sizes and materials of theheater support substrate 40. Generally, when the sizes of the reactormodule 48 and the well 30 are bigger, it takes longer to heat up andcool down the chip. Especially, the thickness of the chip significantlyaffects the thermal cycling. For the heater support substrate 40, if amaterial with low thermal conductivity is used, it takes longer to heatup and cool down the chip when the substrate size is bigger. However,when a material with high thermal conductivity is used, it takes alonger period of time to heat up the chip and takes a shorter period oftime to cool down the chip. If a more powerful heater is available,adopting a bigger support substrate with a high thermal conductivity isencouraged since it can reduce the cooling time.

Chip size is decided based on the simulation results and the fabricationlimits. Though it is desirable to make the chip as small and thin aspossible to shorten the thermal cycling time, some problems occur whenthe chip is made too small. For example, if the chip is designed to betoo thin, some shrinkage may occur while bonding the PDMS layer 34 ontothe glass substrate 36 to form the reactor module 48. Leaks will thenoccur through the gaps caused by the shrinkage. If the chip is toonarrow, it can cause the temperature distribution with the wells 30 tobe less uniform because of the edge effects. An optimal design can onlyachieved by balancing the fabrication process, the chip installation andthe theoretical predictions.

The heating and cooling module was designed and built as a miniaturethermal cycler to provide different temperature levels required for PCR.In one embodiment, the cycler consists of a thin film heating element,such as a Pt micro-film resist heater, for heating, and a fan, such as asmall computer CPU fan blowing from the side, for rapid cooling. A thinfilm heater is sandwiched between two thin metal substrates to form theheating element, and a thermocouple is placed between the top metalsubstrate and thin film heater to control the temperature of the topsubstrate by adjusting the heating power using the feedback informationfrom the thermocouple. The heater sandwich and the thermocouple arebonded together to form a heating unit. The unit is then fixed onto aspecially designed substrate. The reactor module has a size of 22 mm×22mm×1 mm and weighs 0.6 g, as illustrated in FIG. 2. The reactor module48 is placed on the top surface of the heater 38. Four screws are usedto press the four corners of the reactor module to ensure good contactbetween the reactor module and the metal substrate. Alternative types ofcoupling means may be used and are readily understood by a personskilled in the art. The temperature control is accomplished by using acomputer system through a data acquisition card (PCI-DAS 1001,Measurement Computing Corporation, Middleboro, Mass.).

As illustrated in FIG. 7, one embodiment of the system of the presentinvention consists of a reactor module 48, an optical detection module46, and a control module 50. While the control module 50 is illustratedas being in a separate computer, it may be integrated directly into theoptical detection module 46 using the appropriate hardware and softwarecomponents. The reactor module 48 is placed on top of a micro thermalcycler platform 52 which is also integrated into the optical detectionmodule 46. The thermal cycler platform 52 hosts the reactor module 48and provides periodic heating and cooling (fan not shown) to the samplerecipient. The credit-card sized reactor module 48, as described above,is a thin plastic plate with small reaction wells and a glass coverplate to prevent contamination and allow optical detection. The opticaldetector module 46 consists of micro-laser diodes, fiber optics, microsilicon photodiode detectors, optical filters, and an optical switch.The control module 50 contains electronic circuits and micro-chips tocontrol the thermal cycling required for the PCR and to synchronize theoperation sequences of the individual laser diodes, photo-detectors, andthe optical switch, and provides an interface for computer control anddata acquisition. During operation, the wells in the reactor module 48are filled with the test sample and PCR solution. The reactor module 48is coupled with the thermal cycler 52. After the thermal cycler platform52 retreats into the detection unit and the power switch is turned on,the thermal cycling starts and the PCR reaction begins. The opticaldetection module 46 monitors the fluorescent signals in each well andthe laptop computer 50 records the signal intensity. A fan 84 is alsoused in the thermal cycling. The results are analyzed and displayed onthe monitor in real time.

A 4-well miniaturized fluorescence detection system with laser, opticalfiber and optical switch is illustrated in FIG. 8. This detection systemconsists of a laser 54, a filter cube 56, an optical switch 60 and aphoto-detector 58. These four components are connected by opticalfibers. The system is designed to use as few components as possible toreduce the overall size, and is capable of function expansion. The lightfrom the laser 54 is input into a filter cube 56, the laser light isreflected by the filter cube 56 and coupled into the input port of a 1×4optical switch 60. There are 4 ports on the output side of the opticalswitch 60, the input port can be connected to any one of the outputports by a program, controlled by computer 64. The four output fibers ofthe optical switch 60 are mounted above the four wells of the reactormodule 48. They launch excitation light and, in the meantime, collectthe fluorescence emissions from the reaction wells. The collectedfluorescence emissions pass back through the optical switch 60 and thefilter cube 56, and reach the photo-detector 58. Following the detector58 is an electrical operation amplifier 62. Its output is fed to thecomputer 64 which also controls the optical switch 60 and thermal cyclertemperature.

The fiber coupled filter cube 56 is a receptacle style fiber coupledfilter cube. It is similar to a traditional filter cube used in afluorescence microscope. The differences are that (1) there are twofilters inside the cube, (2) three ports are equipped with multimodefibers. There are three filters in traditional microscope filter cubes,which include: exciter (excitation filter), dichroic filter and emitter(emission filter). Since lasers are used as excitation sources insteadof broadband mercury lamps, an excitation filter is not necessary.Lenses are equipped with each of the three ports to collimate and/orfocus the laser or fluorescence beam into/from the fiber. The focalpoint of the three leases are conjugated.

The principle of the fiber coupled filter cube is illustrated in FIG. 9.A dichroic filter 68 vertically reflects laser wavelength and directlytransmits fluorescent wavelength. Laser light is input into the devicefrom “Laser port” 70. It is vertically reflected by the dichroic filter68 and output to the device from “Com port” 72. This laser light excitesthe fluorescence in the sample through the fiber at “Com port” 72 andthe fiber also collects the fluorescence light produced by the sample.The fluorescence light passes through the dichroic filter 68 and finallyreaches the detector. Laser light can also be reflected at the fiber endof the “Com port” 72; about 2% reflected laser light passes through thedichroic filter 68, which represents the noise level of the system. So,a band pass filter 74 is inserted into the “Fluorescent port” 76 toremove this portion of laser light. The dichroic filter 68 and emitterare bought from Chroma Inc, they are especially designed for Cy5 orAlexa Fluor 647™ dye. Filter package with fiber has been done by OZOptics Inc. Fiber connectors 78 are present at each of the Fluorescenceport 76, Laser port 70, and Com port 72. Lenses 80 are used to collimatethe beams.

In order to collect more fluorescent signals (leading to higherdetection sensitivity), 200 μm or 400 μm multimode glass fibers wereconsidered after theoretical calculation and investigation of similaropto-electrical systems were made. Compared with 200 μm fiber, 400 μmfiber can acquire more fluorescence. However, considering the size,mechanical flexibility, compatibility with other components in thesystem, 200 μm fiber was selected for use. Criteria for selecting lasersin this system include high power, small footprint and suitablewavelength. A semiconductor laser is the best choice due to its inherentsmall dimension. The lowest wavelength range of the commercialsemiconductor laser is 630˜650 nm. Thus, Alexa Fluor 647™, one of thelatest, high performance dyes with the highest extinction coefficient,was selected as reporter dye in the real-time PCR, with a peakexcitation wavelength of 650 nm. For the photo detector, compared withother types of detectors such as APD (analog photo-detector), PMT(photo-multiplier tube) and CCD (charge-coupled device), PIN(P-Intrinsic-N), which has the smallest size, was selected. In order toenhance the fluorescence collecting efficiency, minimize the number ofcomponents and reduce the footprint of the optical detection system, afiber-coupled filter cube was designed and developed.

As previously mentioned, during real-time PCR, the optical detectionsystem must monitor the fluorescence intensity in real time. The key isto identify the thermal cycle number at which the reporter dye emissionintensities rise above background noise and start to increaseexponentially. This cycle number is called the threshold cycle, C_(t).The C_(t) is inversely proportional to the number of starting copies ofthe DNA sample in the original PCR solution. Knowing C_(t), the quantityof the DNA to be detected in the sample can be determined.

At least one set of excitation-detection wavelength pair must beavailable at each PCR well to identify both the species in each well. Toincrease the number of samples detected without increasing the number ofwells, additional sources and detectors may be provided. For example, iftwo sources and two detectors are provided, the system can detectdifferent wavelengths being emitted from a common well simultaneously.

As per FIG. 8, the multiplexing is made possible by using an opticalfiber light transmission-switching system, for which switchingcomponents are known to a person skilled in the art. This embodimentrequires two light sources and two detectors. Different excitationlights can be applied to all the wells following the specified sequence.The optical switch allows the emission light from different wells to bemonitored by the filter-detector corresponding to the excitation lightsource according to the specified sequence. This way, the number oflight sources and detectors are independent of the number of wells, andsmaller wells may be employed. In addition, fiber optic technologypermits effectively limitless multiplexing, which permits more reactantwells and more light interactions with little increase ininfrastructure. To create this multiplexing, the switch preferably hasat least as many ports as reaction wells on the side of the opticalswitch in closest communication with the reaction module. For example, afour well reaction module should correspond to an optical switch with atleast four ports on the side of the switch in closest communication withthe reaction module. In additional embodiments, the optical fiber lighttransmission-switching system may include multiple optical switches aswell as switches with a multitude of ports, some ports not always inuse, especially with reaction modules having fewer wells.

In order to operate, the optical detection system must be capable ofswitching the optical paths between wells, and between the specificpairs of the laser diodes and photodiode detectors. This requires thatthe micro-laser diodes, micro-photodiode detectors, optical filters, andoptical switch be synchronized and controlled electronically. Therefore,an electronic device was developed and used for this purpose. Inaddition, the optical detection system must be synchronized with the PCRcontrolling device so that both the PCR and the fluorescent detectionwill operate under the specified sequence. The controlling devices canprovide an interface for computer control and data acquisition.

Simulation and temperature measurement with both thermocouple andRhodamine B dye have shown that the present invention can provide atemperature profile of three different temperature levels required bythe three steps of the PCR (Polymerase chain reaction), such as,denaturation, annealing, and extension steps.

In order to further illustrate the principles and operation of thepresent invention, the following examples are provided. However, theseexamples should not be taken as limiting in any regard.

EXAMPLE 1 Comparative PCR Tests with Reactor Module and MiniatureThermal Cycler (Chip System), and Commercial PCR Machine

Three kinds of DNA template are used to test the chip system. They arehuman genomic DNA(2054), BAC (DJ0416J11) DNA and E. coli O157:H7 DNA.

BAC is an abbreviation of bacterial artificial chromosome. Here,BAC(DJ0416J11) DNA is constructed by insertion of genomic DNA fragmentscorresponding to the genomic DNA amplified in the current experiment,into a vector, which can be replicated in a bacterial host. This hasmany advantages: rapid growth of the host, high stability of the DNAfragment when inside the host, few chimeric clones, easy and rapidpurification of the BAC DNA, and large amounts of sequenced BAC clones.

The different DNA is tested at different protocols, as shown below.TABLE 1 Reagents of PCR mixture for human genomic DNA (2054) ComponentsVolume (μL 1×) DNA(100 ng/μL) 1 10× buffer 2.5 DNTP(2.5 mM) 2.0 MgCl₂0.75 Primer (forward + backward) 1 Taq 0.3 DI-water 17.45

TABLE 2 Reagents of PCR mixture BAC (DJ0416J11) DNA Components Volume(μL 1×) DNA(100 ng/μL) 1 10× buffer 2.5 DNTP(2.5 mM) 2.0 MgCl₂ 0.75Primer (forward + backward) 1 Taq 0.3 DI-water 17.45

TABLE 3 Reagents of PCR mixture for E. coli O157:H7 DNA ComponentsVolume (μL 1×) DNA(2.4 ng/μL and 0.12 ng/μL) 1 10× buffer 2.5 DNTP(2.5mM) 2.0 MgCl₂ 0.75 Primer (forward) 0.3 Primer (backward) 0.3 Taq 0.25DI-water 17.9

For the human genomic DNA (2054) and BAC (DJ0416J11) DNA, the followingthermal condition is used:

-   -   Initial denature: 94° C. for 60 seconds    -   Initial annealing: 59° C. for 30 seconds    -   Initial extension: 72° C. for 30 seconds

35 cycles (30 cycles for J11 DNA) of:

-   -   Denature: 94° C. for 30 seconds    -   Annealing: 59° C. for 30 seconds    -   Extension: 72° C. for 30 seconds

For the E.coli O157:H7 DNA, the following thermal condition is used:

-   -   Initial denature: 95° C. for 10 minutes    -   Initial annealing: 54° C. for 30 seconds    -   Initial extension: 72° C. for 60 seconds

45 cycles of:

-   -   Denature: 94° C. for 20 seconds    -   Annealing: 59° C. for 30 seconds    -   Extension: 72° C. for 60 seconds

At the same time, to compare the amplification results, PCR tests usinga commercial PCR machine are also carried out.

After running PCR tests either with the PCR chip system or with acommercial PCR machine, agarose gel electrophoresis of DNA is conductedto check whether the designed PCR process was successfully achieved. Thedetails of the agarose gel electrophoresis procedure are generally knownto those skilled in the art. Briefly, 0.5% Tris-borate-EDTA (TBE) isused as the buffer, bromophenol blue and xylene cyanol dyes are used asthe tracking dyes, and DNA fragments are visualized by staining withethidium bromide and placing the gel on a ultraviolet transilluminator.For the agarose gel electrophoresis system, FB300 DC power supply(FisherSci, CA) and gel box (Model QSH, international BiotechnologiesInc, USA) are used to run the gel and the Versa Doc imaging system(Bio-Rad Laboratories, USA) is used to take the picture of the gelresults.

The designed PCR chip system can successfully amplify three differentkinds of DNAs: human genomic DNA(2054), BAC (DJ0416J11) DNA and E. coliO157:H7 DNA. For E. coli O157:H7, the primers amplify the stx1 (150 bp)gene of E. coli O157:H7. For the BAC (DJ0416J11) DNA and genomic DNA(2054), the primers are used to amplify the specific (230 bp) gene ofthe human genomic DNA.

FIG. 17 depicts the results observed after running the Agarose gel forthe three amplified DNAs. In FIG. 17, lane 1 is the reference strandgenerated by using PCR marker and other strands are PCR products fromdifferent DNAs and PCR systems. Lane 2 is the PCR product of stx1 (150bp) gene of the E. coli O157:H7 DNA tested on the chip system, lane 3&4are the PCR products of the BAC (DJ0416J11) DNA tested on the chipsystem, lane 5&6 are the products of the genomic DNA(2054) tested on thechip system, and lane 7&8 are the products of the BAC (DJ0416J11) DNA.Lane 5 is the product of genomic DNA on the chip comprising a reactormodule with 1% PVP (polyvinylpyrrolidone) coating, and lane 6 is theproduct of genomic DNA on the chip comprising a reactor module withoutPVP coating. As shown in FIG. 17, for the E. coli O157:H7 DNA, the DNAfragment stx1 (150 bp) is observed at the 150 bp region referring to thePCR marker, and for the BAC (DJ0416J11) DNA and genomic DNA, 230 bpgenes are observed as expected since the primer was designed to amplifythe product of 230 bp gene of the genomic DNA. Both the BAC DNA andgenomic DNA have the same PCR product because the BAC (DJ0416J11) DNA isconstructed with insertion of the same DNA fragment ranges of thegenomic DNA used in this project.

In FIG. 17, the signal of the products of the BAC (DJ0416J11) DNA ismuch stronger than that of the genomic DNA. It is because, in the sameamount of DNA, there are much more of the specific gene fragments in theBAC (DJ0416J11) DNA than occurs in genomic DNA. This means that thereare more initial copies of DNA template strand in the BAC (DJ0416J11)DNA PCR mixtures. Comparing lane 3&4 with lane 7&8, it is shown that forthe BAC (DJ0416J11) DNA, the chip PCR system can generate PCR productsas efficiently as the commercial PCR machine. FIG. 17 also demonstratesthat the signal for the product of the genomic DNA in lane 5 is strongerthan that in lane 6. This implies that the PVP coated reactor moduleresults in a better PCR product than the chips containing PCR reactormodules without this coating.

Difficulties are encountered in amplifying the human genomic DNA in thedesigned chip system. Because the materials of the PCR reactor moduleare different from the material of the commercial PCR tubes, it isnecessary to check the effect of the material of the reactor module onthe PCR process. Our reactor modules were made of PDMS and glass and thecommercial PCR tubes were made of plastic. It is known that glass caninhibit the PCR process for some DNA. Experiments are conducted toverify the effect of glass on the PCR process for the human genomic DNA.

In one experiment, a small piece of glass is placed into the commercialPCR tubes and the PCR reaction is run on the commercial PCR machine withthe human genomic DNA. In those cases, there are no PCR productsobserved when the DNA gel electrophoresis is carried out after the PCRprocess. This implies that the glass inhibits the PCR process of thehuman genomic DNA. Since it is reported that PVP coating could eliminatethe glass inhibition of the PCR process of the genomic DNA [DetlevBelder and Martin Ludwig, Surface Modification in MicrochipElectrophoresis, Electrophoresis, 2003, 24, 3595-3606; Nicole J. Munro,Andreas F. R. Huhmer, and James P. Landers, Robust PolymericMicrochannel Coating for Microchip-Based Analysis of Neat PCR Products,Analytical Chemistry, 2001, 73, 1784-1794] the reactor module and coverglasses are coated with PVP solution, are washed with DI-water, and thePCR test is then conducted with the genomic human DNA.

The PCR products are detected using gel electrophoresis and the resultsare shown in FIG. 18. In FIG. 18, lane 1 is the reference strand whichis generated by using PCR marker, lanes 2 to 6 are the PCR productsobtained from the PCR tests using the reactor modules with PVP coating,and lanes 7 and 8 are the PCR products obtained from using thecommercial PCR machine. It clearly shows that all five tests generatepositive PCR product. Therefore, the PVP coating minimizes thenon-specific adsorption of human genomic DNA on the surface of thereactor module, which is comprised of glass and PDMS.

EXAMPLE 2 Real-Time PCR on a Disposable PDMS Reactor Module with aMiniaturized Thermal Cycler

The reactor module for use in this example comprises two layers of PDMS,bonded onto a 22×22×0.1 mm glass substrate (VWR International). Thebottom layer is for making a reaction well, and the top layer is forholding mineral oil which covers the reactants to prevent evaporation.Liquid Sylgard 184™ (Dow Corning, Michigan, USA) is thoroughly mixedwith curing agent in a weight volume ratio of 15:1. A constant amount ofthe mixture is then poured into a rectangular mold, in order to createPDMS sheets of constant thickness for fabrication of the PCR reactormodules. The thickness of the PDMS sheets is determined by the height ofthe mold and is 0.4 mm in this Example.

After curing for approximately 3 hours at 75° C., the PDMS sheets areremoved from the mold, cut down to the same size as the substrate glass,and a through hole is punched in the center of the PDMS sheet with ametal puncher to form a reaction well. In this example, chips withdifferent well sizes, 3 mm, 2 mm and 1 mm in diameter, are fabricated.The substrate glass of 0.1 mm thickness is coated with a thin layer ofPDMS, because, as previously mentioned, glass is an inhibitor of PCR.The coated glass and PDMS sheet are oxidized in a plasma discharger(PDC-32G, Harrick Scientific, USA) for 60 seconds, and then broughttogether for bonding. Reactor modules with multiple wells are fabricatedsimilarly by punching multiple holes in the PDMS sheets.

To substantially prevent evaporation of the reaction mixture, mineraloil is used to cover the reaction well. Another PDMS sheet withthickness of 0.5 mm is fabricated and is cut down to 10 mm×10 mm. Thethrough-hole in the center is 7 mm in diameter. After a 60 secondsplasma treatment, this sheet is bonded to the PCR chip and centered withthe reaction well to contain the pool of oil.

To demonstrate the concept of multiple PCR wells on a single chip, threeholes of 1 mm diameter are punched on the bottom PDMS sheet of the chip.The center of these three holes forms an equilateral triangle. Thedistance between holes is about 200 μm. Three wells are selected becauseof the limitation of the maximum field of view of our particularfluorescence microscope objective lens. From the heater and chip sizepoint of view, there is no such limitation. FIG. 19 shows schematicallythe assembly of a multiple-well PCR chip.

A specific DNA segment, a 150-bp segment of E. coli O157:H7 stx1 isamplified by using TaqMan™ polymerase. E. coli DNA was extracted fromcells using the protocol and reagents from the QIAGEN Blood and CellCulture DNA Kit. The primer set (Gene Link, Hawthorne, N.Y.) is:forward, 5′-GAC TGC AAA GAC GTA TGT AGA TTC G-3′, and reverse, 5′-ATCTAT CCC TCT GAC ATC AAC TGC-3′. The TaqMan™ probe (Gene Link, Hawthome,N.Y.) is labeled with AlexaFluor 647™ reporter dye and BHQ3™ quencherdye with the following sequence: AlexaFluor 647™ 5′-TGA ATG TCA TTC GCTCTG CAA TAG GTA CTC-3′ BHQ3™. The excitation and emission peaks ofAlexaFluor 647™ are 650 nm and 670 nm, respectively.

Every 100 μl PCR mixture contains 10 μl of 10× buffer, 1.2 μl of eachprimer (25 μM), 2.0 μl of probe (10 μM), 8.0 μl of dNTPs (0.625 mM ofeach), 3.0 μl of MgCl₂, 1.0 μl of TaqMan™ polymerase (5 U/μl) and anappropriate volume of H₂O and DNA template. Each cycle comprises ofthree stages: denaturing at 94° C. for 20 seconds, annealing at 55° C.for 30 seconds, and extension at 72° C. for 30 seconds. Each PCR runbegins with a hot start at 94° C. for 5 minutes, and ends with a finalextension at 72° C. for 10 minutes.

The volumes of the 3 mm, 2 mm and 1 mm diameter reaction wells are 7 μl,3 μl and 0.9 μl respectively. Choosing the 7 μl and 3 μl PCR wells is,on one hand, to prove that the PCR reactor module is flexible and canamplify DNA with different volumes of PCR mixture, and on the otherhand, to allow verification of the PCR results by gel electrophoresiswhich requires a sufficiently large volume. Although real-time PCR isconducted in the experiments and fluorescence detection is done forevery PCR run, gel electrophoresis is conducted to confirm the correctsize of PCR product and no formation of primer dimers. The use of thegel electrophoresis is simply a proof of concept and is not necessaryfor the techniques of the present invention. The well volume of the gelpad is 10 μl so a relatively larger volume of PCR wells is necessary. A2% agarose gel with 0.04% ethidium bromide is used and the results arevisualized with a UV camera (Bio-Rad Gel Doc 1000™, Bio-RadLaboratories, Hercules, Calif.).

The experiments presently described are conducted using a miniaturethermal cycler which was designed and built to provide differenttemperature levels required for PCR, as previously disclosed. The liquidtemperature in the reaction well is lower than that of the substrate,and is calibrated by using a calibration reactor module with anotherthermocouple embedded directly in the reaction well. By adjusting thetemperature of the substrate, the ideal temperature profile in thereaction well can be obtained. FIG. 16 presents the cycling temperaturein the well of a single-well reactor module in the experiment for E.coli O157:H7 stx1 PCR. The ramping-up time from 55° C. to 72° C. andfrom 72° C. to 94° C. is about 6 sec, and 20 sec from 94° C. down to 55°C. The temperature holds 20 sec at 94° C. for denaturing, 30 sec at 72°C. for extension and 30 sec at 55° C. for annealing.

For each PCR run, a newly fabricated reactor module is clamped onto thethermal cycler substrate; PCR mixture is placed into the reaction wellof the PCR reactor module; and the top of the reaction well is coveredwith mineral oil. The thermal cycling program is executed and thefluorescent intensity of the PCR mixture is monitored as the reactionprogresses.

As described above, the developed miniature thermal cycler can bemounted on the stage of any standard fluorescence microscope to measurethe fluorescence intensity for real-time PCR.

Fluorescent images of the sample well are taken by a fluorescencemicroscope (TE2000™, Nikon Inc.) with CCD camera (Qimaging, Vancouver,B.C.) during each PCR run in the experiments. This fluorescentmicroscope is equipped with image analysis software (SimplePCI™) thatallows the calculation of the average fluorescent intensity of anyselected area of the image. Excitation light of 650 nm is providedthrough a 4× microscope objective lens and the image is captured onceevery 6 seconds. Due to the slight temperature dependence of thereporter dye, different intensity levels are observed to occur atdifferent stages of PCR. For the results of this work, the meanintensity during annealing (55° C.) is used to represent thefluorescence intensity of each cycle since the greatest intensity changeoccurs at this temperature level.

Real-time monitoring of PCR can be used for both detection of a specifictype of DNA and quantification of template DNA concentration. To verifythe proposed micro thermal cycler and reactor module system, several PCRexperiments are completed. The experiments use TaqMan™ polymerase chainreaction techniques to amplify the stx1 segment of E. coli O157:H7. Asdescribed above, its length is approximately 150-bp. The resultsdemonstrate that this system can generate the correctly amplified PCRproduct and perform complete real-time PCR detection with differentvolumes of the PCR solution. Additionally, multiple real-time PCRexperiments can be done simultaneously using multiple-well reactormodules.

For single-well reactor modules, real-time PCR tests with differentreaction volumes (7 μl, 3 μl and 1 μl) of reagent mixture and differentinitial DNA concentrations are conducted and the fluorescent intensityat each cycle of PCR is measured. In order to demonstrate that the150-bp stx1 segment could be successfully amplified, real-time PCRexperiments with a 7 μl reaction volume are completed. This relativelylarge reaction volume is required for verification of PCR product sizeusing gel electrophoresis. FIG. 10 shows the mean fluorescent intensityat each cycle, for different amounts of the template DNA (1.3 ng, 2.6 ngand 13.0 ng), and the same mixture volume of 7 μl. For all three amountsof DNA, the characteristic intensity curve is observed, indicating thatthe PCR was carried out successfully. During the initial phase,approximately the first 10 cycles, the intensity remains constant.Following this phase is a rapid increase in fluorescent intensity,followed by a plateau in the intensity level around the 30^(th) cycle.Based on the working principle of the TaqMan™ probe, the reporter dye,AlexaFluor 647™, light is emitted upon cleavage from the BHQ3™ quenchermolecule after reproduction of the specific DNA segment, E. coli O157:H7stx1. Therefore as amplification proceeds, the fluorescent intensityincreases. During the initial phase, although amplification occurred,the change in intensity was below the detection limit. After a certainnumber of cycles, the increase in the fluorescent intensity isdetectable and exponential amplification is observed. Finally, theintensity reaches a plateau as reagents are fully consumed. Comparingthe three cases shown in FIG. 10, it can be clearly seen that themeasured fluorescent intensity starts to increase at a different cyclenumber for different amounts of initial DNA template. Although DNAquantification is not looked at here, the correct trend is shown inthese results: A larger amount of initial template DNA corresponds to anearlier onset of the exponential phase, or an earlier detectableincrease in intensity. The fluorescent intensity starts to increase atapproximately the 15^(th) cycle in the case of 13 ng initial templateDNA, the 18^(th) cycle for the case of 2.6 ng and the 20^(th) cycle forthe case of 1.3 ng.

It is desirable to have a smaller PCR mixture volume for reducing thecost of the reagents and samples and for increasing the number of wellsper unit area of the reactor module. However, using a smaller PCRreaction well must ensure obtaining the correct real-time PCR intensitycurves. FIG. 11 shows the measured fluorescent intensity curves of threetests with the same template DNA concentration, but different volumes (1μL, 3 μL, and 7 μL) of the PCR mixture. As shown in this figure, asimilar trend is present in all three cases, corresponding to thecharacteristic intensity curve described above. This implies thesuccessful PCR in all three mixture volumes. Comparing the three curvesshown in FIG. 11, the measured fluorescent intensity starts to increaseat almost the same cycle number (i.e., the 15^(th) cycle) for all threecases but increases at different rates and reaches a plateau atdifferent intensity levels. In these three cases the fluorescentintensity starts to increase at the same cycle number because they havethe same concentration of initial template DNA. The different rates ofthe intensity increase are due to the lower contribution from the lowertotal of DNA copies and fewer reporter dye molecules associated withsmaller volumes of PCR mixture.

To verify the PCR amplification results, gel electrophoresis is alsoused in this work. Due to the volume limitation, gel electrophoresis isconducted for only the 7 μl and 3 μl samples after each real-time PCRreaction. Typical results are shown in FIG. 12. Since the DNA sample ofE. coli O157:H7 stx1 is 150-bp, the gel results should show a distinctband that corresponds to the 150-bp marker in the PCR ladder. As shownin FIG. 12, the first column is PCR ladder, the second column is the gelelectrophoresis result of a 3 μl PCR reaction, and the third to fifthcolumns are the results of the 7 μl PCR reactions. As expected, thebright band of PCR product for each sample corresponds to the marker'sband at 150-bp. The gel result of the 3 μl sample is not as bright asthe 7 μl samples due to the smaller mixture volume. The gel resultsfurther prove that amplification of the correct PCR product wasachieved. For the 1 μl reaction, the total volume is too small to rungel electrophoresis. However, it is reasonable to assume that the 1 μlcase presented in FIG. 11 has successful PCR amplification, as a similarfluorescent intensity curve is observed as for those of the 7 μl and 3μl cases.

Multiple concurrent reactions can validate the repeatability of the samePCR protocol, or can be used to complete the serial dilution curvesrequired for the quantification of the amount of DNA in the sample in amore efficient manner. Since it is already shown that smaller reactionvolumes, such as 1 μl, could successfully achieve amplification in thepresent system, this small volume is used to carry out multiple-well PCRtests. Three-well reactor modules are tested in this experiment becausethe microscope objective lens we have could only cover an area of threewells, though it is possible to design and fabricate reactor moduleswith more wells for use with alternative detection systems. In theexperiments, a volume of 0.9 μl reaction mixture is applied to each wellin the 3-well reactor module. During each PCR run, the fluorescentintensity of each well is monitored using the fluorescent microscope andthe results are shown in FIG. 13 and FIG. 14. FIG. 13 shows thefluorescent intensity results of three simultaneous reactions that havethe same initial DNA concentration of 0.33 ng/0.9 μl, verifying therepeatability of the PCR protocol and the system. FIG. 14 presents thefluorescent intensity results of three simultaneous reactions that havedifferent initial DNA concentrations. As shown in both FIG. 13 and FIG.14, all intensity curves of the tested cases have the characteristics ofsuccessful PCR amplification. Similar to the results of single wellreactor modules, for the cases with the same initial concentration of atemplate DNA of 0.33 ng/0.9 μl, the measured fluorescent intensitystarts to increase at almost the same cycle number, the 18^(th) cycle,for all three cases shown in FIG. 14. The concentration of 0.33 ng/0.9μl for the curves in FIG. 13 corresponds to the concentration of 2.6ng/7 μl in FIG. 10. The critical cycle numbers for these two samples areindeed the same, i.e., the 18^(th) cycle. Additionally, the fluorescentintensity starts to increase after different cycle numbers for differentinitial amount of template DNA, as shown in FIG. 14. The DNAconcentration of the samples in FIG. 14 is 0.33 ng/0.9 μl, 0.17 ng/0.9μl, and 0.017 ng/0.9 μl, and the critical cycle numbers are the 18^(th),19^(th), and 21^(st), respectively, for the three cases. Comparing theinitial DNA concentration and critical cycle number of PCR runsconducted in the multiple-well reactor module in FIGS. 13 and 14 and thesingle-well reactor module in FIGS. 10 and 11, the results indicate thatthe multiple-well PCR reactor module does not show less efficiency thana single-well reactor module although the volume is reduced. This alsoindicates that the multiple-well PCR is repeatable, and can be used togenerate simultaneous serial dilution curves for quantification.

EXAMPLE 3 PCR Test with Miniaturized Fluorescence Detection System

Using the newly developed laser-optic fiber detection system, real-timedetection of 150 bp E. coli O157:H7 stx 1 gene in our PCR module can beaccomplished. Some preliminary results are shown in FIG. 15. Thefluorescence intensity curves show three phases representing the threecharacteristic phases of the PCR reaction, that is, the flat firstphase, the exponential increase (the second) phase (linear line in Logscale in Y axis), and the saturation phase. FIG. 15 shows the results oftwo PCR runs with different initial DNA concentrations. For DNAconcentrations 12.5 ng/μl and 1.25 ng/μl, the threshold cycle numbers(the starting point of the exponential increase phase) are 19 and 21,respectively. The fluorescence intensity curves are similar to thoseobtained using a commercial real-time PCR machine, indicating ourminiaturized fluorescence detection system works well.

Accordingly, by the practice of the present invention, reaction modules,as well as methods, systems, and devices related to chemical reactions,notably PCR, having heretofore unrecognized characteristics aredescribed.

The disclosures of all cited patents and publications referred to inthis application are incorporated herein by reference.

The above description is intended to enable the person skilled in theart to practice the invention. It is not intended to detail all of thepossible variations and modifications that will become apparent to theskilled worker upon reading the description. It is intended, however,that all such modifications and variations be included within the scopeof the invention that is defined by the following claims. The claims areintended to cover the indicated elements and steps in any arrangement orsequence that is effective to meet the objectives intended for theinvention, unless the context specifically indicates the contrary.

1. A disposable reactor module comprising: a substantiallynon-reflective, thermally conductive substrate; and a layer of polymeron the substrate with the layer of polymer having at least one reactionwell for receiving a fluid sample and with the polymer being chemicallyinert and non-adherent to DNA.
 2. The reactor module of claim 1, whereinthe polymer is poly(dimethylsiloxane) (PDMS).
 3. The reactor module ofclaim 1, further comprising a sealant disposed above at least onereaction well.
 4. The reactor module of claim 1, wherein the substratecomprises glass.
 5. The reactor module of claim 1, wherein the layer ofpolymer has a first groove and a second groove.
 6. The reactor module ofclaim 5, wherein the first groove is an inner groove about the at leastone reaction well, having a diameter of 100 μm to 4 mm, and the secondgroove is an outer groove having a diameter of 100 μm to 4 mm, whereinthe diameter of the second groove is larger than the diameter of thefirst concentric groove.
 7. The reactor module of claim 5, wherein thefirst groove has a depth of from about 50 μm to about 200 μm, and thesecond groove has a depth of from about 50 μm to about 200 μm.
 8. Thereactor module of claim 1 further comprising the at least one reactionwell having an individual maximum volume of from about 0.1 μl to about50 μl.
 9. The reactor module of claim 1 further comprising a peripheralspacer positioned about an opening of at least one reaction well. 10.The reactor module of claim 9, wherein the peripheral spacer has aheight of from about 50 μm to about 500 μm.
 11. A method for real-timemonitoring of a temperature-controlled chemical reaction involvingfluorescence emissions, the method comprising the steps of: a) providingat least one fluid sample in a disposable reactor module comprising asubstrate and a layer of polymer on the substrate, with the layer ofpolymer having at least one reaction well for receiving the sample; b)sealing at least one reaction well; c) heating and cooling the reactormodule to allow a chemical reaction to occur; d) directing excitationwavelengths to the sample to create fluorescence emissions; e) capturingthe fluorescence emissions; and f) processing the fluorescence emissionsto monitor the chemical reaction.
 12. The method of claim 11, whereinthe at least one fluid sample has a volume of from about 0.1 μl to about50 μl.
 13. The method of claim 11, wherein the chemical reaction is aPolymerase Chain Reaction (PCR).
 14. A miniature multiplex fluorescencedetection system for detecting fluorescence emissions from at least onesample on a reactor module having a plurality of reaction wells, thesystem comprising: at least one light source coupled to the reactionwells, for generating light at excitation wavelengths; at least onedetector for receiving detection wavelengths from the reaction wells;and an optical switching device, coupled between the detector and thereaction wells, to direct emissions of fluorescence to the detector. 15.The system of claim 14, further comprising a filter for directing lightfrom at least one light source to the optical switching device and fordirecting the fluorescence emissions to at least one detector, whereinthe light from the at least one light source is directed to the reactionwells by the optical switching device.
 16. The system of claim 14wherein at least one light source is a semiconductor laser.
 17. Thesystem of claim 14 wherein at least one detector is a PIN photodetector.
 18. The system of claim 14, further comprising a heating andcooling module coupled to the reactor module for modulating atemperature of the sample.
 19. The system of claim 14, furthercomprising a housing for hosting the light source, detector, switchingdevice, heating and cooling module, and a stage for coupling to thereactor module, wherein a slot is provided in the housing for insertingthe reactor module therein.
 20. A device for real-time monitoring of atemperature-controlled chemical reaction involving fluorescenceemission-detection, the device comprising: a multiplex fluorescencedetection system for detecting fluorescence emissions from fluid samplescontained in reaction wells, the system comprising: at least one lightsource coupled to the reaction wells, for generating light at excitationwavelengths; at least one detector for receiving detection wavelengthsfrom the reaction wells; and an optical switching device, coupledbetween the detector and the reaction wells, to direct emissions offluorescence to the detector; a heating and cooling module formodulating a temperature of the samples; and a control module forcontrolling the fluorescence detection system and monitoring thechemical reaction by processing the fluorescence emissions.
 21. Thedevice of claim 20, further comprising a disposable reactor modulecomprising a substrate and a layer of polymer on the substrate, with thelayer of polymer having the reaction wells for receiving fluid samples.22. The system of claim 21 wherein the polymer is poly(dimethylsiloxane)(PDMS).
 23. The system of claim 21 wherein the layer of polymer has aplurality of grooves around the reaction wells.
 24. The device of claim20 further comprising a sealant for reducing evaporation of the fluidsamples contained in the reaction wells out of the reaction wells. 25.The device of claim 20, further comprising a filter for directing lightfrom at least one light source to the optical switching device anddirecting the fluorescence emissions to at least one detector, whereinthe light from at least one light source is directed to the reactionwells by the optical switching device.
 26. The device of claim 20,wherein at least one light source is a semiconductor laser.
 27. Thedevice of claim 20 wherein at least one detector is a PIN photodetector.
 28. The device of claim 21 further comprising a housing forhosting the light source, detector, switching device, heating andcooling module, and stage, and wherein a slot is provided in the housingfor inserting the reactor module therein.
 29. The device of claim 20wherein the control module is integrated within the fluorescencedetection system.
 30. The device of claim 20, wherein the device isportable.
 31. The device of claim 20, wherein the chemical reaction is aPolymerase Chain Reaction (PCR).